CN102384907B - Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content - Google Patents

Method by utilize vanillin-sulfuric acid colorimetry to measure glabridin content Download PDF

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CN102384907B
CN102384907B CN 201110230297 CN201110230297A CN102384907B CN 102384907 B CN102384907 B CN 102384907B CN 201110230297 CN201110230297 CN 201110230297 CN 201110230297 A CN201110230297 A CN 201110230297A CN 102384907 B CN102384907 B CN 102384907B
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glabridin
concentration
solution
standard
linear regression
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CN102384907A (en
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樊金玲
张麦茹
朱文学
李欣
唐浩国
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Henan University of Science and Technology
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Henan University of Science and Technology
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Abstract

The invention relates to a method by utilizing vanillin-sulfuric acid colorimetry to measure glabridin content. Collection process of chromogenic reagent absorbency value comprises the steps that: vanillin methanol solution with concentration of 20mg/mL is prepared, sulfuric acid methanol solution with concentration in percentage by volume of 30 percent is prepared, glabridin standard solution with concentration of 0.5mg/mL is prepared, glabridin standard test solution with range of the concentration of 0.01 to 0.5mg/mL is prepared, the absorbency value is collected, the chromogenic reagent is stable and reliable within 60 minutes under the room temperature, so the collection of the absorbency value information of the chromogenic reagent is guaranteed, and finally a linear regression equation is established. Being proved by a recovery test and a repeatability test: the method is stable to analyze, the repeatability conforms to the requirement, the method is proved to be completely reliable, the linear regression equation can provide a quantitative standard to the measurement of the content of the glabridin to be tested, and a test result and test precision, which are identical to the high-efficient liquid-phase spectrum, can be obtained.

Description

Vanillin-sulfuric colorimetry is measured the method for glabridin content
Technical field
The invention belongs to plant content determination techniques field, refer more particularly to the method that a kind of vanillin-sulfuric colorimetry is measured glabridin content.
Background technology
Glycyrrhiza glabra ( Glycyrrhiza glabraL .) in the glabridin that contains of institute spy ( Glabridin) belonging to the isoflavan class, glabridin has diversified pharmaceutically active and receives much concern.The molecular formula of glabridin is C 20H 20O 4, molecular weight is 324.37.
There is data to show, glabridin can be used to do the adjuvant in the whitening cosmetics, by the activity of restraint of tyrosinase and dopachrome interconvertible enzyme TRP-2, hinders 5, the polymerization of 6-dihydroxy indole DHI stops melanic formation, thereby reaches the effect of skin whitening.
Also have data to show that glabridin has following characteristic:
(1) have stronger antioxidation activity, it is close with vitamin E to remove the oxygen radical ability;
(2) have antibiotic, anti-born of the same parents poison, anti-breast cancer cel l proliferation, and significantly hypotensive, reducing blood lipid and stronger neuroprotective effect, its application prospect is very wide.
What present mode about analysis glabridin content adopted is high performance liquid chromatography, and the employed high performance liquid chromatograph of high performance liquid chromatography is expensive, and testing cost is high, and detection time is longer, and is also very high to operating personnel's requirement.
Although colourimetry is to measure at present content of material quantitative analysis method comparatively commonly used, yet under the vanillin-sulfuric acid condition, use the method for colorimetric method for determining glabridin content so far there are no report.
Summary of the invention
For addressing the above problem, the invention provides a kind of vanillin-sulfuric colorimetry and measure the method for glabridin content, the method is divided into two large steps, first step is the gatherer process of nitrite ion absorbance information, second step is that the equation of linear regression of glabridin standard detection solution concentration-mean light absorbency value is set up process, the required time of the method shortens greatly than high performance liquid chromatography, and nitrite ion is reliable and stable in room temperature 60min, is fit to very much the centralized detecting of a large amount of glabridin content to be measured.
For achieving the above object, the present invention adopts following technical scheme:
A kind of vanillin-sulfuric colorimetry is measured the method for glabridin content, and the method is mainly concerned with: the standard items purity HPLC of glabridin 〉=98%, absolute methanol, mass percent concentration is 98% sulfuric acid, vanillic aldehyde, thermostat water bath, cuvette, ultraviolet-visible pectrophotometer; The method has resorcinol structure and C ring C by the B ring 2, C 3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration, the recycling ultraviolet-visible pectrophotometer is measured the absorbance of nitrite ion under its maximum absorption wavelength 534nm, thereby finish the gatherer process of nitrite ion absorbance information: under described acid condition, the shade of nitrite ion and the content of glabridin are remarkable linear positive correlation relation, meet Lambert-Beer's law, thereby for the equation of linear regression foundation of glabridin standard detection solution concentration-mean light absorbency value is laid a good foundation, described equation of linear regression provides quantitative criterion for the mensuration of glabridin content in the later testing sample.
The gatherer process of described nitrite ion absorbance information is as follows:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker, in beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde, vanillic aldehyde methanol solution after the dissolving is transferred in the first volumetric flask of 100 mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the vanillic aldehyde methanol solution after the rinse also transferred in the first volumetric flask, add in the first volumetric flask till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again.
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and the second volumetric flask that places 100 mL, adds the absolute methanol of 70 mL in the second volumetric flask again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution.
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker, in beaker, add a small amount of absolute methanol with the standard items of dissolving glabridin, the standard items methanol solution of glabridin after the dissolving is placed the 3rd volumetric flask of 25mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, add in the 3rd volumetric flask till absolute methanol solution to the 25 mL scale, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL again.
4. prepare 14 parts of concentration ranges at the glabridin standard detection solution of 0.01~0.5 mg/mL
The glabridin standard solution is 14 parts in drawing respectively 3., the uptake of described 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL, and place respectively 14 5 mL volumetric flasks, add successively in 14 5 mL volumetric flasks again till absolute methanol to the 5 mL scale, obtain 14 parts of concentration after shaking up and be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution of mg/mL and 0.5 mg/mL, or referred to as the glabridin standard detection solution of 14 parts of concentration ranges at 0.01~0.5 mg/mL.
5. gather absorbance
In the glabridin standard detection solution of 0.01~0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4., then to the tool plug test tube of described 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be the vanillic aldehyde methanol solution of 20 mg/mL and 2.5 mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution, then the tool plug test tube of described 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min, each tool plug test tube obtains the clarification nitrite ion of 5.5 mL, obtains altogether 14 parts of described nitrite ions; The described nitrite ion (namely extracting the described nitrite ion less than 5.5 ∕ 3mL) that extracts less than 1 ∕ 3 from each tool plug test tube removes the rinse cuvette, utilize ultraviolet-visible pectrophotometer to gather the absorbance information of described nitrite ion, using the absorbance information that obtains 14 different numerical value of 14 parts of described nitrite ions in the same cuvette situation like this, the absorbance information of these 14 different numerical value is called one group, does altogether under the same conditions three groups of parallel experiments to obtain three groups of absorbance information; Three absorbances that the described nitrite ion of same concentrations in three groups is gathered carry out arithmetic mean, namely obtain the mean light absorbency value of three groups of described nitrite ions of same concentrations, can access in this way the mean light absorbency value of the described chromophoric solution of other concentration in three groups, obtain altogether 14 mean light absorbency values, described 14 mean light absorbency values are corresponding at the glabridin standard detection solution phase of 0.01~0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4..
It is as follows that the equation of linear regression of described glabridin standard detection solution concentration-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution concentration of mg/mL and 0.5 mg/mL is the x axle, take above-mentioned 5. described in 14 mean light absorbency values set up the equation of linear regression of glabridin standard detection solution concentration-mean light absorbency value as the y axle, represented by y=4.4483x and y=1.4208x+0.4013 by the described equation of linear regression of the calculating of EXECL software, wherein y=4.4483x is that glabridin standard detection solution is 0.01 mg/mL in its concentration, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 the equation of linear regression when mg/mL and 0.14 mg/mL, linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The equation of linear regression that y=1.4208x+0.4013 is glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL, linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when described boundary flex point.
Because adopt aforesaid technical scheme, the present invention has following superiority:
1, the relative high performance liquid chromatography of the present invention have equally the result accurately, the precision advantages of higher, replaced valuable high performance liquid chromatograph, so that testing cost significantly reduces.
2, the present invention is based on the B ring and have resorcinol structure and C ring C 2, C 3It is the glabridin of singly-bound reacted and generated the different red complex of the depth under acid condition from vanillic aldehyde reagent chromophoric solution, chromophoric solution has the maximum light absorption rate under the 534nm wavelength, utilize the ultraviolet-visible pectrophotometer collection to measure the absorbance of chromophoric solution, meet Lambert-Beer's law, set up on this basis the equation of linear regression of glabridin content-absorbance.
3, method of the present invention is easy and simple to handle, quick, finishing whole detection operating process only needs less than one hour, greatly shorten detection time, and nitrite ion is reliable and stable in room temperature 60min, thereby be fit to very much the centralized detecting to a large amount of glabridin content to be measured, be particularly suitable for the detection of dynamic of each production phase in industrial abstract and the refining glabridin process.
Description of drawings
Fig. 1 is glabridin standard detection solution concentration-mean light absorbency value typical curve.
Embodiment
Vanillic aldehyde claim again vanillic aldehyde ( Vanillin).Document announcement is arranged, and vanillic aldehyde is the qualitative Identification and determination analysis of Chinese crude drug developer commonly used, and this mechanism is based on: C encircles C 2, C 3Between be singly-bound and have resorcinol or the flavone compound of phloroglucin A-ring structure, can with the vanillic aldehyde reagent generation red complex that reacts.
The B ring of glabridin has C in resorcinol structure and the C ring 2, C 3Singly-bound, thereby glabridin and the vanillic aldehyde generation red complex that under acid condition, also can develop the color, this is for the invention provides basic condition.Although basic condition is set up, the sensitivity of chromogenic reaction is relevant with the concentration of acid in the reaction system, and large its sensitivity of the low then moisture of acid concentration is just poor.Through single factor experiment, it is that 98% the concentrated sulphuric acid is as the acid condition of reaction system that the present invention has selected mass percent concentration, greatly improved sensitivity and the stability of chromogenic reaction, thereby and mass percent concentration is that 36.6% concentrated hydrochloric acid is because contained humidity causes more greatly the sensitivity of chromogenic reaction greatly to reduce improper this reaction system that is used as.
The standard items of the used glabridin of the present invention are buied from market, the standard items purity HPLC of used glabridin 〉=98%.
The present invention is the method that a kind of vanillin-sulfuric colorimetry is measured glabridin content, and the method has resorcinol structure and C ring C by the B ring 2, C 3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration, the recycling ultraviolet-visible pectrophotometer is measured the absorbance of nitrite ion under its maximum absorption wavelength 534nm, thereby finish the gatherer process of nitrite ion absorbance information: under described acid condition, the shade of nitrite ion and the content of glabridin are remarkable linear positive correlation relation, meet Lambert-Beer's law, thereby for the equation of linear regression foundation of glabridin standard detection solution concentration-mean light absorbency value is laid a good foundation, described equation of linear regression provides quantitative criterion for the mensuration of glabridin content in the later testing sample.
The gatherer process of nitrite ion absorbance information of the present invention is as follows:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker, in beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde, vanillic aldehyde methanol solution after the dissolving is transferred in the first volumetric flask of 100 mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the vanillic aldehyde methanol solution after the rinse also transferred in the first volumetric flask, add in the first volumetric flask till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again.
Attention: the addition of described a small amount of absolute methanol solution is not necessarily equal several times 1., but the total addition of absolute methanol in 1. must be strict controlled in 100 mL.
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and the second volumetric flask that places 100 mL, adds the absolute methanol of 70 mL in the second volumetric flask again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution.
Concentration of volume percent provides sour environment for 30% sulfuric acid methanol solution for chromogenic reaction.
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker, in beaker, add a small amount of absolute methanol with the standard items of dissolving glabridin, the standard items methanol solution of glabridin after the dissolving is placed the 3rd volumetric flask of 25mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, add in the 3rd volumetric flask till absolute methanol solution to the 25 mL scale, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL again.
Attention: the addition of described a small amount of absolute methanol is not necessarily equal several times 3., but the total addition of absolute methanol in 3. must be strict controlled in 25 mL.
4. prepare 14 parts of concentration ranges at the glabridin standard detection solution of 0.01~0.5 mg/mL
The glabridin standard solution is 14 parts in drawing respectively 3., the uptake of described 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL, and place respectively 14 5 mL volumetric flasks, add successively in 14 5 mL volumetric flasks again till absolute methanol to the 5 mL scale, obtain 14 parts of concentration after shaking up and be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution of mg/mL and 0.5 mg/mL, or referred to as the glabridin standard detection solution of 14 parts of concentration ranges at 0.01~0.5 mg/mL.
Above-mentioned 4. in 14 parts of concentration ranges of reply make not isolabeling at the glabridin standard detection solution of 0.01~0.5 mg/mL, or to 14 5 mL volumetric flasks make not isolabeling also can, in case chaotic.
5. gather absorbance
In the glabridin standard detection solution of 0.01~0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4., then to the tool plug test tube of described 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be the vanillic aldehyde methanol solution of 20 mg/mL and 2.5 mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution, then the tool plug test tube of described 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min, each tool plug test tube obtains the clarification nitrite ion of 5.5 mL, obtains altogether 14 parts of described nitrite ions; The described nitrite ion that extracts less than 1 ∕ 3 from each tool plug test tube removes the rinse cuvette, utilize ultraviolet-visible pectrophotometer to gather the absorbance information of described nitrite ion, using the absorbance information that obtains 14 different numerical value of 14 parts of described nitrite ions in the same cuvette situation like this, the absorbance information of these 14 different numerical value is called one group, does altogether under the same conditions three groups of parallel experiments to obtain three groups of absorbance information; Three absorbances that the described nitrite ion of same concentrations in three groups is gathered carry out arithmetic mean, namely obtain the mean light absorbency value of three groups of described nitrite ions of same concentrations, can access in this way the mean light absorbency value of the described chromophoric solution of other concentration in three groups, obtain altogether 14 mean light absorbency values, described 14 mean light absorbency values are corresponding at the glabridin standard detection solution phase of 0.01~0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4..
Above-mentioned cuvette must use same, wants this cuvette of Rapid Cleaning after absorbance information of every collection, and the absorbance that only gathers under same cuvette and same ultraviolet-visible pectrophotometer is Reduce measurement error to greatest extent.
The model that above-mentioned ultraviolet-visible pectrophotometer uses is the UV-2800 type, the absorbance that it can the described chromophoric solution of Accurate Measurement.
Described nitrite ion is reliable and stable in room temperature 60min, and this provides assurance for the absorbance information that gathers described nitrite ion.
It is as follows that the equation of linear regression of glabridin standard detection solution concentration of the present invention-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution concentration of mg/mL and 0.5 mg/mL is the x axle, take above-mentioned 5. described in 14 mean light absorbency values set up the equation of linear regression of glabridin standard detection solution concentration-mean light absorbency value as the y axle, represented by y=4.4483x and y=1.4208x+0.4013 by the described equation of linear regression of the calculating of EXECL software, wherein y=4.4483x is that glabridin standard detection solution is 0.01 mg/mL in its concentration, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 the equation of linear regression when mg/mL and 0.14 mg/mL, linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The y=1.4208x+0.4013 equation of linear regression that is glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL wherein, linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when described boundary flex point.
Y=4.4483x and y=1.4208x+0.4013 provide quantitative criterion for the mensuration of glabridin content in the later testing sample.
Fig. 1 is the glabridin standard detection solution concentration set up according to described equation of linear regression-mean light absorbency value typical curve, horizontal ordinate is that the x axle is the concentration range of glabridin standard detection solution among the figure, and concentration among the figure (mg/mL) represents the concentration of glabridin standard detection solution; Ordinate is that the y axle is the scope of mean light absorbency value among the figure, and A represents the mean light absorbency value among the figure.
The application example of described equation of linear regression
From the glabridin subtractive process, randomly draw its sample A and sample B, sample A is become sample A solution to be measured by above-mentioned 3. arrangements of steps, and then utilize the concentration of described equation of linear regression calculation sample A solution to be measured.
The sample A solution to be measured of 0.5 mL is joined in the tool plug test tube of 10 mL, adding successively 2.5 mL concentration in the tool plug test tube is that 20 mg/mL vanillic aldehyde methanol solutions and 2.5 mL concentration of volume percent are 30% sulfuric acid methanol solution, tool plug test tube is carried out capping and rocks mixing, then take out after tool plug test tube being placed 20 ℃ of water-bath insulation 20 min, obtain the clarification nitrite ion of sample A solution to be measured.
The described nitrite ion that extracts less than 1 ∕ 3 from above-mentioned tool plug test tube removes the rinse cuvette, utilize ultraviolet-visible pectrophotometer to gather the absorbance information of described nitrite ion, do altogether under the same conditions three groups of parallel experiments to obtain the absorbance information of three groups of different numerical value.The absorbance of three groups of different numerical value is carried out arithmetic mean, obtain the mean light absorbency value of the described nitrite ion of sample A solution to be measured, with mean light absorbency value substitution equation of linear regression, the concentration that calculates glabridin in the sample A solution to be measured is 0.124 mg/mL.
The concentration that can calculate equally glabridin in the sample B solution to be measured according to aforesaid way is 0.221 mg/mL.
Recovery experiment
1. recovery experiment 1
Respectively get 5mL concentration and be six parts of the sample A solution to be measured of 0.124 mg/mL and place respectively six 10 mL test tubes, in six 10 mL test tubes, add successively glabridin standard solution 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL that concentration is 0.5 mg/mL again, six 10 mL test tubes are made six addition marks, then in six test tubes of making mark, add respectively absolute methanol to 10mL scale and end, fully obtain the sample A solution of six parts of dilutions behind the mixing.
The former content value of glabridin is 0.124mg/mL * 5mL=0.62 mg in the above-mentioned six duplicate samples A solution to be measured, and the adding value of glabridin is respectively 0 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg and 0.5 mg in the glabridin standard solution.
Get each 0.5mL of sample A solution of described six parts of dilutions, by above-mentioned 5. step and can calculate respectively glabridin concentration in the sample A solution of described six parts of dilutions according to described equation of linear regression, and calculating total measured value of the glabridin in the sample A solution of described six parts of dilutions, total measured value is respectively 0.620 mg, 0.721 mg, 0.819 mg, 0.92 mg, 1.022 mg and 1.121 mg.
Deduct the measured value that described former content value is glabridin addition in the glabridin standard solution with described total measured value, the ratio of described measured value and adding value is the recovery, obtain standard deviation between the described recovery value with EXECL software, the ratio of described standard deviation and recovery arithmetic mean is exactly the relative standard deviation of recovery test.
Concrete outcome sees following table for details:
2. recovery test 2
Can make as stated above the recovery test of sample B solution to be measured.Concrete outcome sees the following form:
The reappearance test
Reappearance test 1:
Respectively get 0.5mL concentration and be eight parts of the sample A solution to be measured of 0.124 mg/mL and place respectively eight 10 mL tool plug test tubes, by above-mentioned 5. step and can calculate respectively the measured value of glabridin concentration in the described eight duplicate samples A solution to be measured according to described equation of linear regression, try to achieve eight standard deviations between the measured value with EXECL software, the ratio of described standard deviation and measured value arithmetic mean is exactly the relative standard deviation of reappearance test, and described relative standard deviation is 1.72%.
Reappearance test 2:
Record as stated above the measured value of glabridin concentration in the eight duplicate samples B solution to be measured, and finally to try to achieve relative standard deviation be 1.24%.
The concrete outcome of above-mentioned reappearance test 1 and 2 is summed up and is seen the following form:
Show that by concrete example application and recovery test and reappearance test the testing sample of randomly drawing is by the inventive method, according to the implementation step, all can Accurate Determining go out the content of glabridin in the testing sample.The presentation of results of recovery experiment and reappearance experiment methods analyst of the present invention stablize, reappearance meets the requirements, prove that method of the present invention is fully reliably, described equation of linear regression provides quantitative criterion for the mensuration of glabridin content in the later testing sample.
In addition in order to verify reliability of the present invention, by high performance liquid chromatography method of the present invention is verified, net result shows: method of the present invention has accuracy, and method of the present invention can obtain the testing result identical with high performance liquid chromatography and precision.
Need to prove at last:
1, the whole course of reaction of the present invention should be in anhydrous state;
2, operating process of the present invention is fast and convenient, the result is accurate, and the described chromophoric solution that obtains is reliable and stable in 60min;
3, equation of linear regression is at the present invention's lower gained that imposes a condition, and is equal to replacement according to method of the present invention and all should be encompassed among the technical scheme of the present invention.

Claims (1)

1. a vanillin-sulfuric colorimetry is measured the method for glabridin content, and the method is mainly concerned with: the standard items purity HPLC of glabridin 〉=98%, absolute methanol, mass percent concentration is 98% sulfuric acid, vanillic aldehyde, thermostat water bath, cuvette, ultraviolet-visible pectrophotometer; The method has resorcinol structure and C ring C by the B ring 2, C 3It is the glabridin of singly-bound and vanillic aldehyde reagent reacts and be to make the reactant liquor colour developing under 98% the sulfuric acid condition at mass percent concentration, the recycling ultraviolet-visible pectrophotometer is measured the absorbance of nitrite ion under its maximum absorption wavelength 534nm, thereby finish the gatherer process of nitrite ion absorbance information: under described acid condition, the shade of nitrite ion and the content of glabridin are remarkable linear positive correlation relation, meet Lambert-Beer's law, thereby for the equation of linear regression foundation of glabridin standard detection solution concentration-mean light absorbency value is laid a good foundation, described equation of linear regression provides quantitative criterion for the mensuration of glabridin content in the later testing sample; It is characterized in that:
The gatherer process of described nitrite ion absorbance information is as follows:
1. compound concentration is the vanillic aldehyde methanol solution of 20 mg/mL
Take by weighing the vanillic aldehyde of 2.0g with beaker, in beaker, add a small amount of absolute methanol with the dissolving vanillic aldehyde, vanillic aldehyde methanol solution after the dissolving is transferred in the first volumetric flask of 100 mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the vanillic aldehyde methanol solution after the rinse also transferred in the first volumetric flask, add in the first volumetric flask till absolute methanol solution to the 100 mL scale, obtaining concentration after shaking up is the vanillic aldehyde methanol solution of 20 mg/mL again;
2. the dose volume percent concentration is 30% sulfuric acid methanol solution
Accurately measuring 30 mL mass percent concentrations with transfer pipet is 98% sulfuric acid and the second volumetric flask that places 100 mL, adds the absolute methanol of 70 mL in the second volumetric flask again, obtains concentration of volume percent after shaking up and be 30% sulfuric acid methanol solution;
3. compound concentration is the glabridin standard solution of 0.5 mg/mL
Take by weighing the standard items of 12.5 mg glabridins with beaker, in beaker, add a small amount of absolute methanol with the standard items of dissolving glabridin, the standard items methanol solution of glabridin after the dissolving is placed the 3rd volumetric flask of 25mL, use again a small amount of absolute methanol rinse beaker 2-3 time, and the standard items methanol solution of glabridin after the rinse also transferred in the 3rd volumetric flask, add in the 3rd volumetric flask till absolute methanol solution to the 25 mL scale, obtaining concentration after shaking up is the glabridin standard solution of 0.5 mg/mL again;
4. prepare 14 parts of concentration ranges at the glabridin standard detection solution of 0.01~0.5 mg/mL
The glabridin standard solution is 14 parts in drawing respectively 3., the uptake of described 14 part of 0.5 mg/mL glabridin standard solution is followed successively by 0.1 mL, 0.3 mL, 0.5 mL, 0.7 mL, 0.9 mL, 1.1 mL, 1.3 mL, 1.4 mL, 2.0 mL, 2.6 mL, 3.2 mL, 3.8 mL, 4.4 mL and 5.0 mL, and place respectively 14 5 mL volumetric flasks, add successively in 14 5 mL volumetric flasks again till absolute methanol to the 5 mL scale, obtain 14 parts of concentration after shaking up and be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution of mg/mL and 0.5 mg/mL, or referred to as the glabridin standard detection solution of 14 parts of concentration ranges at 0.01~0.5 mg/mL;
5. gather absorbance
In the glabridin standard detection solution of 0.01~0.5 mg/mL, respectively draw 0.5 mL and join respectively in the tool plug test tube of 14 10 mL from above-mentioned 14 parts of concentration ranges that obtain 4., then to the tool plug test tube of described 14 10 mL add respectively 2.5 mL above-mentioned 1. in the concentration of preparation be the vanillic aldehyde methanol solution of 20 mg/mL and 2.5 mL above-mentioned 2. in the concentration of volume percent of preparation be 30% sulfuric acid methanol solution, then the tool plug test tube of described 14 10 mL is carried out capping and rocks mixing being placed in 20 ℃ of water-baths and taking out behind insulation 20 min, each tool plug test tube obtains the clarification nitrite ion of 5.5 mL, obtains altogether 14 parts of described nitrite ions; The described nitrite ion that extracts less than 1 ∕ 3 from each tool plug test tube removes the rinse cuvette, utilize ultraviolet-visible pectrophotometer to gather the absorbance information of described nitrite ion, using the absorbance information that obtains 14 different numerical value of 14 parts of described nitrite ions in the same cuvette situation like this, the absorbance information of these 14 different numerical value is called one group, does altogether under the same conditions three groups of parallel experiments to obtain three groups of absorbance information; Three absorbances that the described nitrite ion of same concentrations in three groups is gathered carry out arithmetic mean, namely obtain the mean light absorbency value of three groups of described nitrite ions of same concentrations, can access in this way the mean light absorbency value of the described chromophoric solution of other concentration in three groups, obtain altogether 14 mean light absorbency values, described 14 mean light absorbency values are corresponding at the glabridin standard detection solution phase of 0.01~0.5 mg/mL with above-mentioned 14 parts of concentration ranges that obtain in 4.;
It is as follows that the equation of linear regression of described glabridin standard detection solution concentration-mean light absorbency value is set up process:
6. with above-mentioned 4. described in 14 parts of concentration be followed successively by 0.01 mg/mL, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 mg/mL, 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 the glabridin standard detection solution concentration of mg/mL and 0.5 mg/mL is the x axle, take above-mentioned 5. described in 14 mean light absorbency values set up the equation of linear regression of glabridin standard detection solution concentration-mean light absorbency value as the y axle, represented by y=4.4483x and y=1.4208x+0.4013 by the described equation of linear regression of the calculating of EXECL software, wherein y=4.4483x is that glabridin standard detection solution is 0.01 mg/mL in its concentration, 0.03 mg/mL, 0.05 mg/mL, 0.07 mg/mL, 0.09 mg/mL, 0.11 mg/mL, 0.13 the equation of linear regression when mg/mL and 0.14 mg/mL, linear concentration range: (0.01-0.14) mg/mL, coefficient R 2=0.9999; The equation of linear regression that y=1.4208x+0.4013 is glabridin standard detection solution when its concentration is 0.14 mg/mL, 0.2 mg/mL, 0.26 mg/mL, 0.32 mg/mL, 0.38 mg/mL, 0.44 mg/mL and 0.5 mg/mL, linear concentration range: (0.14-0.5) mg/mL, coefficient R 2=0.9957; Can find out: x=0.14 mg/mL is the boundary flex point of y=4.4483x and y=1.4208x+0.4013, and two linearity curves have Different Slope when described boundary flex point.
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